The present invention relates to the on-line detection of properties of the effluent of column chromatography that require appreciable time for measurement, and more particularly to an assay of radioactivity with enhanced sensitivity and resolution.
In column chromatography, such as gas-liquid, gas-solid, liquid-liquid or liquid-solid chromatography, detection is generally accomplished by passing the column effluent through or past a detector which senses and records over time the rises and falls of a property of the effluent related to the concentration of the compounds of interest. Both identification and quantification of components of the mixture being separated depend on accurate recording of these concentration changes and the time at which they occur. In order to achieve and preserve maximal resolution, detector volumes are generally minimized and flow rates are adjusted for maximal response to rapid changes in concentration. Only small portions of the effluent are present in the detector at any given time, and the time each portion remains in the detector is minimized.
When radioactivity is to be measured, however, maximizing resolution limits sensitivity. Because of the random nature of radioactive decay, the precision of any radioassay is a function of the number of radioactive events that contribute to the measurement. In radiochromatography, each portion of the effluent must remain in the detector sufficiently long for the required number of radioactive emissions to be detected. Detector volumes and flow rates are compromises between these opposing requirements. In current high performance (or pressure) liquid chromatography (HPLC), for example, time in the detector can often be extended up to about 20 seconds without intolerable degradation of resolution, even though in other detectors much shorter times are generally thought desirable. If the sample contains less radioactivity than can be measured with acceptable precision in 20 sec., flow-through detection is generally not useful. The alternative is to fractionate the effluent for subsequent radioassay of each fraction for much longer times. To achieve the same resolution, separate fractions must be collected at least every 20 sec. Many fractions must then be assayed, and this can occupy a counting device for many hours. For this reason, most workers find flow-through detectors more convenient to use and reserve fraction-collecting for those assays involving very low levels of radioactivity and either limit the time each fraction is counted or sacrifice resolution for sensitivity by collecting fewer fractions.
Baba et al. described delivering a stream of HPLC effluent mixed with scintillation fluid, but without segmentation of the stream to achieve discrete fractionation, through five scintillation counters in series. S. Baba, et al., J. Chromatograph, 244 (1982) 57 and 392 (1987) 157. They recorded the predicted increase in precision over that from a single detector but also noted appreciable and predictable loss of resolution, manifested by an increase in peak width from the first to the fifth detector, caused by mixing of one part of the effluent with the next. This effect is particularly troublesome with detergent-containing scintillation fluids which tend to wet even polytetrafluoroethylene (PTFE) tubing.
Accordingly, it is an object of the present invention to provide a method for the on-line assay of a property of a stream which requires appreciable time for sensitive measurement, which method combines high sensitivity and high resolution.
It is also an object to provide such a method wherein the stream is a column chromatographic effluent.
Another object is to provide such a method wherein the assay is a radioassay.
A further object is to provide such a method which is simple, easy, and economical.